The present invention relates to the field of functional analysis of cotton genes on a genomic scale. More specifically, the present invention relates to a method for high-throughput functional analysis of cotton genes on a genomic scale using virus-induced gene silencing (VIGS). The present invention also relates to a transient expression vector for transiently expressing genes in cotton plants and to a method for transient expression of genes in cotton plants.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the Bibliography.
Cotton (Gossypium spp.) is the world's most important fiber plant and a significant oilseed crop, being grown in more than 80 countries with a record of 122 million 480-pound bales in world production during the 2006/2007 growing season (United States Department of Agriculture-Foreign Agricultural Service). The deficit between consumption and production has happened in 1994/1995 and is forecasted to continue to widen to 2.5 million 480-pound bales in the 2009/2010 growing season (United States Department of Agriculture-Foreign Agricultural Service [USDA-FAS] 2009). Cotton production provides income for approximately 100 million families, and approximately 150 countries are involved in cotton import and export (Lee et al., 2007). Its economic impact is estimated to be approximately $500 billion/year worldwide. Moreover, modifying cotton-seed for food and feed could profoundly enhance the nutrition and livelihoods of millions of people in food-challenged economies. Cotton is also a potential candidate plant of renewable biofuel. Cotton fiber is composed of nearly pure cellulose. Compared to lignin, cellulose is easily convertible to biofuels. Optimized cotton fiber production and processing will ensure that this natural renewable product will be competitive with petroleum-derived synthetic non-renewable fiber to ensure more sustainable development.
To solve the issues stated above, many agronomic properties of cotton, such as fiber length and strength, agricultural productivity, drought tolerance and pest resistance need to be enhanced by the availability of genetic resources and rapid methods to identify gene functions (Udall et al., 2006).
Cotton is an important crop that is widely grown and is used to produce both natural textile fiber and cotton seed oil. Cotton fiber is a model system for the study of cell elongation and cell wall and cellulose biosynthesis. And it is unicellular, therefore cell elongation can be evaluated independently from cell division. One of the most significant benefits for using cotton fiber as a model system for plant development is that a culture method for cotton ovules was perfected by Beasley and Ting (1973).
Gossypium includes approximately 45 diploid (2n=2x=26) and five tetraploid (2n=4x=52) species, all exhibiting disomic patterns of inheritance. Most modern cotton varieties are forms of Gossypium hirsutum (upland cotton, tetraploid), about 95% of annual cotton crop world wide, although three other species are also utilized to a lesser extent, Gossypium barbadense (tetraploid), Gossypium arboreum (diploid), and Gossypium herbaceum (diploid). These three species are also very important genetic resources and offer gene reservoir for special breeding purpose. For example, G. herbaceum, with high resistance to biotic and abiotic stresses, can be used as a good start genetic material for interspecies crossings with G. hirsutum to improve its resistance to various stresses. Therefore, a species independent method for gene functional analysis in Gossypium genus and relative plants is also greatly needed.
Currently, complete sequencing of cotton genomes is just beginning. Meanwhile, an ever-expanding set of Gossypium EST sequences (about 400,000 now) and derived unigene sets from different libraries constructed from a variety of tissues and organs under a range of growth conditions are accessible on the web, as well as by microarray analyses based on these sequences (Udall et al., 2006). The availability of other plant genomic sequences serves as a useful platform for identifying and annotating putative orthologs in cotton EST databases. Even though analogies can be drawn between cotton fiber differentiation and the formation of leaf trichomes and secondary-walled xylem cells, ultimately the function of putative orthologous genes needs to be tested directly in cotton. In addition, cotton fibers are known to express genes with no known homologs in other plants, which may confer some of the unique properties of fibers. Even if the function of several transcriptional factors genes have been tested in Arabidopsis, their exact functions need to be further verified in the homologus cotton plant.
An important strategy to identify agronomic and quality traits of Gossypium is by stable transformation into cotton. However, the inefficient production of stably transformed cotton plants limits gene identification on a large scale. Moreover, such procedure is laborious and time consuming and not suitable for high throughput analysis on a genomic scale. Furthermore, only few cultivars can be used for host for transformation. Normally, it is difficult to directly identify important genetic elements from good start genetic materials by stable transformation in cotton.
Thus, it is desired to develop a method for the species independent high-throughput functional analysis of Gossypium genes on a genomic scale.